Heat Shock Protein-22 and Rev Block In Astrocytes

星形胶质细胞中的热休克蛋白 22 和 Rev 阻断

• 批准号: 7418538
• 负责人: RAGHAVENDAR R THIPPARTHI
• 金额: $ 7.53万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-30 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7418538
• 关键词: AIDS Dementia Complex; Acquired Immunodeficiency Syndrome; Affect; Antiviral Agents; Astrocytes; Binding; Biochemical; Biological Assay; Brain; Cells; Cytoplasmic Protein; Data; Development; Environment; Fluorescence Microscopy; Gene Expression; Genetic Transcription; Goals; HIV; HIV-1; Health; Heat shock proteins; Highly Active Antiretroviral Therapy; Human; In Vitro; Infection; Knowledge; Lead; Light; Mediating; Metabolism; Methodology; Microglia; Microscopic; Molecular; Molecular Chaperones; Nature; Nuclear; Outcome; Patients; Personal Satisfaction; Pharmaceutical Preparations; Post-Transcriptional Regulation; Prevention; Production; Protein Family; Public Health; RNA; RNA Interference; Range; Research; Role; SRC-associated p68 protein; Severities; Small Interfering RNA; Study Section; Testing; Therapeutic; Treatment Protocols; Viral Proteins; Virus Latency; Work; base; cell type; design; experience; in vivo; innovation; mRNA Export; macrophage; novel; nucleocytoplasmic transport; therapeutic vaccine; trafficking; uptake; viral RNA

DESCRIPTION (provided by applicant): The ineptness of highly active anti-retroviral therapy (HAART) regimen against HIV-associated dementia patients (HAD) necessitates continuous development of new drugs against brain infections. The long-term goal is to develop an anti-HIV therapeutic strategy using inducible chaperone family proteins, specifically small heat shock proteins. The objective of this particular application is to determine the role of heat shock protein-22 (Hsp22) in the post-transcriptional regulation of HIV-1 gene expression. The central hypothesis of this application is that Hsp22 interferes with the nuclear uptake of Rev, thus impairing Rev function and subsequent HIV-1 production in astrocytes. The hypothesis has been formulated from the preliminary data, showing that Hsp22 is expressed at high levels in astrocytes and inhibits Rev function. The rationale for the proposed project is that, once the mechanism of how Hsp22 inhibits Rev function is known, using new and innovative strategies, its production can be either up- or down-regulated pharmacologically to the prevention and treatment of AIDS, including HAD. Guided by preliminary data, the central hypothesis will be tested to accomplish the objective of this application by pursuing the following two specific aims: 1. Determine the role of Hsp22 in the post-transcriptional regulation of HIV-1 gene expression. The working hypothesis is that Hsp22 interacts with Rev and interferes with its function by perturbing its intracellular localization, thus resulting in inhibition of HIV-1 production in target cells. Under this aim, using biochemical approaches, such as in vitro and in vivo binding assays, the physical interaction between Hsp22 and Rev will be tested. Furthermore, using Northern analysis the functional relevance of these interactions will be determined. 2. Determine the role of Hsp22 in HIV-1 latency in astrocytes. The working hypothesis of this aim is that high levels of Hsp22 in astrocytes contributes to the cytoplasmic predominance of Rev, which in turn will interfere with the nuclear uptake of Rev, and subsequent inhibition of HIV-1 production. Under this aim, well proven RNA interference (siRNA) approach will be used to inhibit the expression of Hsp22 in astrocytes, after which Rev localization will be examined by immuno-fluorescence microscopy and Rev-mediated RRE-RNA export will be analyzed by Northern analysis. The proposed work is innovative, because no cytoplasmic protein(s), specifically Hsp22, was previously shown to participate in the post-transcriptional regulation of HIV-1 gene expression. The research proposed in this application is significant because it is expected to provide the knowledge needed to develop pharmacologic strategies to eliminate the HIV-1 reservoirs or to maintain the viral latency eternally. In either scenario, cellular activities that disrupt the nucleocytoplasmic transport of viral RNAs are expected to become valuable antiviral weapons in combating the HIV-1 infection. Project Narrative: The proposed research is relevant to public health, because, understanding the role of Hsp22 will shed light on the molecular basis of HIV-1 latency operating at the level of RNA metabolism in CNS-based cell type and would lead to novel HSP-based therapeutic and vaccine approaches for the control of HIV-1 infection. One of the advantages of Hsp22 over viral proteins is that the antiviral affect will be broadly effective against all HIV-1 isolates and clades, including the CNS strains. Thus, the results are expected to be applicable to the health of human beings.

描述（由申请人提供）：针对HIV相关痴呆患者（HAD）的高效抗逆转录病毒治疗（HAART）方案的无效性需要持续开发针对脑感染的新药。长期目标是开发一种使用诱导型伴侣蛋白家族蛋白，特别是小的热休克蛋白的抗HIV治疗策略。本申请的目的是确定热休克蛋白-22（Hsp 22）在HIV-1基因表达的转录后调节中的作用。本申请的中心假设是Hsp 22干扰Rev的核摄取，从而损害Rev功能和随后星形胶质细胞中的HIV-1产生。该假设已制定从初步的数据，表明热休克蛋白22在星形胶质细胞中的高水平表达，抑制Rev功能。该项目的基本原理是，一旦已知Hsp 22如何抑制Rev功能的机制，使用新的和创新的策略，其生产可以上调或下调，以预防和治疗艾滋病，包括HAD。在初步数据的指导下，将通过追求以下两个具体目标来测试中心假设以实现本申请的目标：1.确定Hsp 22在HIV-1基因表达的转录后调节中的作用。工作假设是Hsp 22与Rev相互作用，并通过干扰其细胞内定位来干扰其功能，从而导致靶细胞中HIV-1产生的抑制。在这一目标下，使用生物化学方法，如体外和体内结合试验，将测试Hsp 22和Rev之间的物理相互作用。此外，使用北方分析，这些相互作用的功能相关性将被确定。2.确定Hsp 22在星形胶质细胞中HIV-1潜伏期中的作用。该目标的工作假设是星形胶质细胞中高水平的Hsp 22有助于Rev的细胞质优势，这反过来又会干扰Rev的核摄取，以及随后对HIV-1产生的抑制。在此目标下，将使用经证实的RNA干扰（siRNA）方法来抑制星形胶质细胞中Hsp 22的表达，之后将通过免疫荧光显微镜检查Rev定位，并通过北方分析来分析Rev介导的RRE-RNA输出。这项工作是创新的，因为没有细胞质蛋白（S），特别是热休克蛋白22，以前被证明参与HIV-1基因表达的转录后调控。本申请中提出的研究具有重要意义，因为它有望提供开发消除HIV-1储库或永久保持病毒潜伏期的药理学策略所需的知识。在这两种情况下，破坏病毒RNA的核质转运的细胞活动有望成为对抗HIV-1感染的有价值的抗病毒武器。项目叙述：拟议的研究与公共卫生有关，因为了解Hsp 22的作用将揭示HIV-1潜伏期在CNS细胞类型中RNA代谢水平上的分子基础，并将导致新的基于HSP的治疗和疫苗方法用于控制HIV-1感染。Hsp 22相对于病毒蛋白的优势之一是抗病毒作用将对所有HIV-1分离株和进化枝（包括CNS株）广泛有效。因此，该结果有望适用于人类健康。